Online

Carroll, Brittany L.

Cellular, Molecular, and Biomedical Sciences Graduate Program

2019

Ph. D.

Cells synthesize proteins, the molecular instruments of all cellular processes, via intermediate biomolecules that are susceptible to damage at every step. Known as the central dogma of molecular biology, genes encoded in deoxyribonucleic acid (DNA) are transcribed, spliced, and matured into messenger ribonucleic acid (mRNA). These nucleic acids direct protein synthesis by the pairing of nucleotide triplets with transfer RNA (tRNA). tRNAs concomitantly decode the so-called codon, as they escort the correct amino acid to the ribosome for extension of the nascent polypeptide chain. Damage to any of these intermediate biomolecules can be highly damaging to protein synthesis, leading to aberrant biochemical processes, aging, cancer, or apoptosis. Accordingly, cells have evolved essential response and repair pathways to ensure that replication, transcription, and translation occur with high fidelity. In this dissertation, we interrogate two enzymes involved in these quality-control measures: 1) a DNA glycosylase which recognizes damage to the DNA bases, and 2) a tRNAHis guanylyltransferase-like protein (or THG1-like proteins, TLPs) which repairs truncated or mismatched tRNA via 3' to 5' polymerization. DNA is assaulted daily to the tune of 30,000 lesions per cell per day by exogenous and endogenous stressors. One of many DNA repair pathways, the base excision repair (BER) pathway, removes the small non-bulky, and oxidized DNA lesions from the genome. DNA glycosylases are the first enzymes in the concerted mechanism tasked with scanning the entire genome for DNA damage and initiating the repair of lesions. The human genome encodes 11 DNA glycosylases, which possess overlapping substrate specificities within BER. The DNA glycosylase, endonuclease three (Nth), recognizes and removes oxidized pyrimidines during all phases of the cell cycle. We have solved the first X-ray crystal structure of human Nth-Like 1 (hNTHL1), which revealed a novel open conformation. This unprecedented example of an Nth DNA glycosylase undergoing interdomain rearrangement provides important insight into the molecular mechanism of this critical guardian of the genome. In eukaryotes, tRNAs must be modified at the 5' end during maturation. tRNAHis guanylyltransferase (THG1), an essential gene in yeast, catalyzes the addition of guanine to the 5' end of tRNAHis. Reverse polymerization requires adenylation (or guanylation) to activate the 5' end of the tRNA. After adenylation, there is a shift of the 5'-phosphate of the tRNA to accommodate the forthcoming nucleophilic attack by the 3'-OH of the incoming nucleotide. In contrast to their human counterparts, the archaeal TLP enzymes utilize the 3' to 5' NTP-polymerization reaction to repair 5'-degraded tRNA molecules. We have solved the first crystal structure of a TLP caught in an intermediate step following activation by guanylation, showing that the base rotates within the nucleotide binding site to align the active site.